Compositions comprising protocatechuic acid and methods of use

ABSTRACT

This disclosure is directed to methods of using compositions comprising protocatechuic acid (PCA) to kill virus including a Covid-19 virus. The compositions generally comprise protocatechuic acid, a liquid vehicle, and a stabilizer. In embodiments, the liquid vehicle comprises an alcohol and/or water. In preferred embodiments, the oil is an essential oil. In embodiments, the compositions may comprise principally protocatechuic acid, liquid vehicle, and stabilizer as the main ingredients. The compositions may be sprayed onto various products and articles of manufacture, and mammalian and human skin, to kill virus including a Covid-19 virus and to further protect the product or human skin for a period of time up to 24 hours or more. In preferred embodiments, an alcohol component kills virus on contact, and after drying, the residual surface PCA coating provides a continuing anti-viral function.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/947,256 filed Jul. 24, 2020, which is herein incorporated byreference in its entirety.

BACKGROUND OF THE DISCLOSURE Field of the Invention

This disclosure is generally directed to compositions comprisingprotocatechuic acid and methods of using compositions comprisingprotocatechuic acid (PCA) to inactivate COVID-19 virus SARS CoV-2 and tointerrupt its disease transmission, provide a treatment and enhance thetranslation to clinical application.

Description of the Related Art

The COVID-19 virus SARS CoV-2 has brought on an international pandemic.The World Health Organization named this new coronavirus SARS-CoV-2, andthe disease, COVID-19 (CoronaVirus Disease-19). Corona viruses arecommon and have been recognized for many years. People around the worldcommonly get infected with human coronaviruses: 229E, NL63, OC43, andHKU1. However, it has become apparent that the SARS-CoV-2 is unlike anyother coronavirus; i.e; MERS, SARS-CoV, and the common cold. SARS-COV-2is unique clinically in that it is highly contagious. It rapidly becamea pandemic and it is very aggressive. It affects not only therespiratory system but there are pathological manifestations in theskin, brain, liver and kidney as well

SARS-CoV-2 differs in molecular structure from other coronavirus. It isknown that prion—like domains exist with a distribution in thecoronavirus that are unlike prion-like domains in other viruses andwhich may play important functional roles in transmission. Theintroduction of the prion to the molecular structure of the SARS-CoV-2may require a different and unique therapeutic reagent or method. Prionis a term first used to describe the mysterious infectious agentresponsible for several neurodegenerative diseases found in mammals,including Creutzfeldt-Jakob disease (CJD) in humans. The word itselfderives from ‘proteinaceous infectious particle’ and it refers to theinitially heretical hypothesis that the infectious agent causing thosediseases consists only of protein with no nucleic acid genome. Allpreviously known pathogens contain nucleic acids, which enable them toreproduce. The prion hypothesis explained why the mysterious infectiousagent is resistant to ultraviolet radiation, which breaks down nucleicacids, but is susceptible to substances that disrupt proteins.

The clinical manifestations of SARS Co-2 are unique and differ fromother respiratory virus. SARS Co-2 is very contagious. At the same timethe clinical manifestation may vary from no symptoms, mild symptoms andfor some very severe symptoms and or death. Although the primarypathology is in the respiratory system (a lung infection), this newvirus has new and expanded clinical manifestations beyond prior coronalviral observations including pulmonary modes. In addition, there havebeen heart, skin, and brain manifestations with this virus. Its clinicalmanifestations may also include a severe inflammatory component called a“cytokine storm” in the lungs.

There is also thrombosis in other areas of the body beyond the lungsthat has resulted in amputation of the limbs. The numbers of thoseinfected and those dying is large.

COVID-19 is thus a pathological and clinical enigma. The is no knownprophylactic drug. There is no specific therapeutic drug. There is nodrug or treatment with the present prospect of translation to clinicalpractice. The present scientific therapeutic measures are limited totesting and tracking after which recommendations can be made for socialdistancing and face masks. Quarantines have been used. State and localgovernments have mandated non-essential business shut downs and limitedpublic gatherings. These are after the fact measures and none aretherapeutic. Testing or tracking of an entire population is notrealistic and, in any case, false positives and negatives exist with alltesting. Add to that that the pre-symptomatic patient is a likely causeof transmission before they have recognizable symptoms. In addition, itis recognized that there are certain subjects, perhaps 20% of those withthe disease that are super spreaders yet are only identified after thefact. The fact remains that COVID-19 is not well understood and ishighly contagious.

While waiting for an FDA approved drug and a vaccine, the humanitarianand societal costs in morbidity and mortality climb. The pandemic israpidly evolving and there is every expectation it will continue sincethe epidemiology of this new strain is yet to be established. Eventhough its identification was established, the virus has spreadglobally, causing thousands of deaths and having an enormous impact oninternational health systems and economies. Testing is being developedand expanded. However, the asymptomatic have not been included in thetesting protocol so a definitive understanding of SARS-CoV-2epidemiology is lacking. The data is yet to be sufficient to rule outthe false positives since those that had a cold in the past had thecoronavirus. The false negatives are yet to be defined.

The only present means of containing this very contagious virus is bymitigation. There is no known established treatment. Prevention iscurrently limited to the use of sanitizers and disinfectants. Sanitizerstypically use alcohol which is only bactericidal or viricidal when wet.Upon drying there is no further anti-microbial effect. The dry surfaceis vulnerable to another microbial residence. In addition, there is asubstantial lack of knowledge and misuse of disinfectants in the generalpopulation. Traditional disinfectants may use antimicrobials whichfunction chemically or biochemically. Their biochemical inter-actiondisrupts the viral interaction with the host and/or physically disruptsthe virus prongs or wall.

Crystals themselves provide a therapeutic application by their physicalnature. They have known cytotoxic properties which can achieve atherapeutic result. It is known that the physical properties of crystalscan have an antimicrobial property independent or in conjunction withtheir biochemical properties. Their many sharp edges have the potentialto physically disrupt a microbe's integrity.

The coronaviruses are particularly physically vulnerable. The coveringof the coronavirus is surrounded by many projections like a crown. Theprojections are called prongs or spikes. These spikes are the virulentcontact agent with the host cell. They penetrate the human cell and theinfection is then propagated. The spikes and underlying thin wallcoating of the viral body are vulnerable to physical disruption.Physical disruption is one potential method of stopping the cellularinvasion and the clinical disease. Crystals have a physical structurethat is irregular, rough, and sharp with potential to physically disrupta microbes' spikes and covering.

Accordingly, there is a need and an opportunity for prevention andtreatment to expand beyond or in conjunction with the chemical methodsto one that is physical disruption and one that is effective against inspite of various prion mutations. The use of crystals presents thepossibility of creating an antiviral agent capable of protecting from ordestroying SARS-CoV-2.

Protocatechuic acid crystals like other crystals are typically observedand considered only in the dry state. However, it known that PCA retainsvarious crystalline shapes while in a liquid medium. The PCA crystal wasfirst reported in liquid to be in three different forms in 1949. Thefollowing publication from 1949 is extensively illustrated. Seehttps://royalsocietypublishing.org/doi/10.1098/rspa.1949.0064 RobertWilliams Wood. Published:22 Jun. 1949.https://doi.org/10.1098/rspa.1949.0064.

In 1983, Agmon, et al supported Wood's work and showed that somecrystalline shapes were stable in form and other were rapidly changingin liquid. See Agmon I, Herbstein F H, Thomas J M. Spontaneousdeformation of protocatechuic acid monohydrate crystals:crystallographic aspects. Proc. R. Soc. Lond. 1983. A387311-330.http://doi.org/10.1098/rspa.1983.0062.

SUMMARY OF THE INVENTION

This disclosure is generally directed to compositions comprisingprotocatechuic acid and methods of using compositions comprisingprotocatechuic acid (PCA) to inactivate COVID-19 virus SARS CoV-2 and tointerrupt its disease transmission, provide a treatment and enhance thetranslation to clinical application. The compositions generally compriseprotocatechuic acid in the dry state and or in liquid solution orenvironment.

Examples that closely replicate the clinical environment demonstratethat PCA was virucidal when physically engaging the SARS CoV2 virus inan aqueous medium are provided below.

Interruption of Transmission: Based upon these findings there isapplication for interruption of transmission in many ways. There is theapplication for sanitizing human skin and disinfecting personalprotective equipment; e.g., face masks. Spraying the dual reagents onhard surfaces kills what is present and leave a PCA residue coating tobe viricidal. The interrupts the transmission from hard surfaces tohands to the subject's face. In addition, fogging and or aerophizationof trains, plane and automobiles. These methods of interruption may beused in buildings and or arenas. It may be applied to room filters.

Treatment: The present clinical significance of these observations isthat PCA in fluid would retain its various crystalline shapes andtherefore its anti-viral properties lend PCA to a treatment modality.This would be expected in an aerosol and body fluids. The application isavailable for oral, intravenous and intraperitoneal routes. It may beused in a nebulizer or ventilator.

Translation to Clinical Practice: PCA is FDA designated as safe with aGenerally Recognized As Safe for food substance flavoring. PCA is safeto ingest and therefore ready for translation to clinical application.Its ready availability and low cost of goods insures a wide societaldistribution.

The virus is physically disrupted when it comes in physical contact withprotocatechuic acid crystals and therefore renders the viruspathologically inactivated. PCA and related methods interrupts thetransmission of the coronavirus in several ways. PCA crystals be appliedtopically and delivered in a liquid vehicle solution. Alcohol provides adual effect when it is the vehicle for use as a sanitizer and ordisinfectant on skin or hard surfaces. The alcohol kills what is presentand after drying the residual surface PCA coating provides a continuinganti-viral function. It may be applied to hard surfaces to protect fromthis mode of transmission. It may be used as a coating and onapplication transmission is interrupted by PCA; in one embodiment it maybe in a liquid vehicle, and a stabilizer.

In embodiments, the liquid vehicle comprises an alcohol and thestabilizer comprises an oil. In preferred embodiments, the oil is anessential oil. In embodiments, the compositions may comprise principallyprotocatechuic acid, liquid vehicle, and stabilizer as the mainingredients. In preferred embodiments, the compositions may compriseonly protocatechuic acid, a liquid vehicle, and a stabilizer.

Water is the vehicle for interruption and or treatment of the mucousmembranes of the nose, mouth, pharynx, respiratory tree and lungs. Thecompositions may be sprayed or fogged onto various products and articlesof manufacture, and mammalian and human skin, to kill and protect fromviruses including the Covid 19 SARS-Co-2 virus.

In preferred embodiments, the liquid vehicle includes water or a lowboiling point alcohol and/or alcohol combination ranging from about 0°C. to about 100° C. The alcohol may include methanol and/or butanol, butpreferably ethanol and/or propanol for human use.

Low boiling point alcohols can include alcohols such as methanol,ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,2-methyl-1-propanol, 2-methyl-2-propanol, 1-pentanol,3-methyl-1-butanol, 2,2-dimethyl-1-propanol, and cyclopentanol.

An essential oil is generally a concentrated hydrophobic liquidcontaining volatile (easily evaporated at normal temperatures) chemicalcompounds extracted from a plant. Essential oils are also known asvolatile oils, ethereal oils, aetherolea, or as the oil of the plantfrom which they were extracted. In preferred embodiments, the essentialoil may comprise orange, mint, peppermint, cedarwood, lemon, southernblue gum, evergreen, clove, and/or spearmint.

Additional possible ingredients include glycerin, propylene glycol,sodium benzoate, as well as sanitizers and disinfectants. Propyleneglycol may be a vehicle for topical application and or foraerophilization. Seehttps://ajph.aphapublications.org/doi/pdf/10.2105/AJPH.34.5.477.

One embodiment of the present disclosure provides for the spraying ofprotocatechuic acid (PCA) in solution on a variety of articles. Upondrying the result being a coating PCA on and or in the articlessubstance. The preferred embodiments are an article of manufactureincluding personal protective equipment (PPE). Personal protectiveequipment includes caps, hats, and other head coverings, gowns, masksand facemasks, gloves, shoes, and other footwear, etc. The applicationmay be at the time of manufacture and or after-market application in theclinical setting.

The products also include hard surfaces that are frequently touched orhandled by the public. This would include, as nonlimiting examples, ATMmachines and credit card payment devices, gas pump handles, doors anddoorknobs, tables and counters in public spaces including restaurants,etc.

In embodiments, a single spray with dual action can kill virusesincluding SARS-CoV-2. The first action includes an alcohol effect beforeevaporation. This kills the virus on contact and then dries quicklyleaving a coating of PCA. The PCA coating presence on the articlemaintains a presence to continually kill bacteria and viruses uponsubsequent contact. PCA in this application thus acts as a dual phaseinhibitor.

In preferred embodiments, the virus is SARS Co 2. The alcohol will killSARS Co 2 on contact and then the PCA coating will continue to kill andprotect the surface, or article of manufacture, the subject's skin, fromSARS Co 2 for a significant period of time up to at least 24 hours ormore.

PCA may be used in a variety of treatments. In preferred aerosolizedembodiments, the virus is SARS Co 2 is the target. An aerosolized fogwith the PCA in suspension will kill SARS Co 2 on contact. Subsequently,the PCA coating will continue to kill and protect the surface, orarticle of manufacture, the subject, from SARS Co 2 for a significantperiod of time up to at least 24 hours or more.

In preferred oral ingestion embodiments the ready absorption places thePCA crystal in solution in the plasma which will kill SARS Co 2 oncontact. This will last throughout the known presence of PCA in themammalian body for several days. The PCA known metabolism is that it issubsequently found in urine and feces intact and as subsequentmetabolites.

In the preferred embodiments of intraperitoneal route of application,the ready absorption places the PCA crystal in solution in the plasmawhich will kill SARS Co 2 on contact. This will last throughout theknown presence of PCA in the mammalian body for several days. The PCAknown metabolism is that it is subsequently found in urine and fecesintact and as subsequent metabolites.

In the preferred embodiments of intravenous route of application, thepresence places the PCA crystal in solution in the plasma which willkill SARS Co 2 on contact. This will last throughout the known presenceof PCA in the mammalian body for several days. The PCA known metabolismis that it is subsequently found in urine and feces intact and assubsequent metabolites.

In preferred embodiments of a PCA coating application to room andrespiratory filters and facilities, the PCA crystal in the ventilationsystem such that viruses are reduced or destroyed on contact.

In the preferred embodiments of the PCA coating application with anebulizer places the PCA crystals in the subject's respiratory systemsuch that pathologic microbes are reduced or destroyed on contact.

Also disclosed is a method of treating pathological condition caused bySARS CoV2 virus, comprising the coating of skin, oral cavity, nares,nasopharynx, and pulmonary tree.

Also disclosed is methods of treatment of the pathological conditioncaused by the SARS CoV2 virus comprising the intravenous andintraperitoneal route.

In embodiments, the present disclosure provides multiple routes oftherapeutic delivery of PCA; e.g., coating of various products includingpersonal protective equipment and hard surfaces. Normal size crystals(177 um) may be delivered by the oral route. Smaller sized crystals mayalso be used for intravenous, intraperitoneal and aerosol delivery to apatient, subject (prophylactic) or facility.

Other features and aspects will be apparent from the following detaileddescription, the drawings, and the claims.

DETAILED DESCRIPTION

The following detailed description is provided to assist the reader ingaining a comprehensive understanding of the methods, products, and/orsystems, described herein. However, various changes, modifications, andequivalents of the methods, products, and/or systems described hereinwill be apparent to an ordinary skilled artisan.

Protocatechuic acid (PCA) is a phytochemical, a powerful antioxidant,which is common in nature. It is found in abundance in plants, treeleaves, and edible fruits and vegetables. PCA is naturally present inthe ground, rivers, ponds, and lakes. It is manufactured by bacteria inthe soil. It is present in the human diet. The metabolism in nature andthe human body is known. After ingestion, injection or a topicalapplication, the tissue and organs of residence are known. The humanbowel bacteria manufacture PCA. The routes of elimination are known.There are no known human toxic effects of PCA. PCA is non-allergenic anda non-inflammatory. It is also non-mutagenic. PCA's health benefits areestablished in the literature and validated by supporting evidence toprior US patents. Importantly for a new therapeutic, protocatechuic acid(PCA) has been designated as Generally Recognized As Safe (GRAS) by theFDA as a flavoring substance. PCA may be biochemically manufacturedand/or extracted from plants in an amorphous or crystalline state. Bothstates have the anti-viral properties of low pH, anti-protease, dockingblocker and hormonal and cellular immunity remains inherent in themolecule.

As mentioned, protocatechuic acid (PCA) is a compound with powerfulanti-inflammatory properties. One of the ways the human body responds toPCA is with a massive anti-inflammatory response. The pathologicaleffect seen in clinical cases of SARS Co-2 has been termed a ‘cytokinestorm’ in the lungs. PCA is a powerful anti-inflammatory by itsanti-catabolic cytokine blocker properties. PCA acts as a tyrosinaseinhibitor in other applications. PCA is also a protease inhibitor. PCAhas anti-viral docking properties.

Protocatechuic acid (PCA) is thus a broad-spectrum antiviral destroyingantibiotic when coating of cloth and/or metal surfaces. PCA has thephysical properties of a crystal with sharp protrusions that may disruptthe coating of microbes in the dry state or in solution.

Protocatechuic acid (PCA) has viricidal effect on the SARS CoV2 virus asproven by the Examples provided below by a method design that replicatedthe clinical environment.

SARS CoV-2 virus retains its viability and pathogenesis in aqueousmedium; in animals, humans and in air borne droplets. Outside of thisaqueous environment SARS CoV-2 virus has an extremely limited viability.When droplets with SARS CoV-2 virus come to rest on hard surfaces,thereby subject to drying the life of the virus is just hours.

Therefore, the interruption of transmission and or treatment will be inan aqueous environment. The interruption of transmission must bedesigned to engage the coronavirus in such an environment; i.e.aerophilized droplets. The successful treatment will likely be likewise;within the confines of the mammalian body's nasopharynx, bronchi, lungsand vascular system.

The supporting Examples were performed in a liquid environment,replicating the clinical environment. The reagent, protocatechuic acid(PCA) was applied to an article simulating the clinical condition ofspraying the article with a dual purpose. The alcohol vehicle replicatedthe lethal effect on contact of an existing virus on a hard surface. Theresidual PCA coating provided a lasting antiviral effect for subsequentviral exposure. The subsequent droplet containing virus engaged the PCAdrug in an aqueous environment. There was a virucidal effect in 10minutes of on the SARS CoV2 virus upon engaging the PCA crystal.Therefore, this Example substantiated the interruption of transmissionfor this drug; application to PPE, skin sanitizer, and as a disinfectantused in facility misting or fogging.

The Example design also replicated the therapeutic potential for PCA asall drug/viral engagements are in an aqueous medium. The Example designdelivered the SARS CoV2 virus in the traditional droplet vehicle, henceupon contact with the PCA coated article converted the environment fromdry to wet. This replicated the clinical environment of the nasopharynx,bronchi, lungs, and blood. The PCA crystals may be delivered to thoseanatomical locations by several known routes; oral, intravenous, andintra-peritoneal. In addition, the topical application is possible byfilters in airway systems, nebulizer, or ventilators.

The translation to clinical practice is facilitated by PCA existing FDAdesignation as generally recognized as safe; it is edible. PCA isreadily available in large quantities and the cost of goods is low.

The crystal structure of a reagent may cause physical disruption ofviral coating resulting in viral death. PCA in the blood will retain itscrystalline and therefore its anti-viral properties. The crystallinephysical nature may be constantly changing in solution. PCA in a liquid(water and possibly alcohol or other vehicles) retains variouscrystalline forms of varying shapes, but with sharp edges. Thiscrystalline antiviral factor of PCA is present whether in a liquidvehicle or dried on a hard surface.

Mutations of a COVID-19 virus would require a generally differingtherapeutic, biochemical, or chemical approach. PCA may involve physicaldisruption of the COVID-19 virus and low pH and so it would be expectedto work against any mutations.

In embodiments, the size of PCA crystals may be approximately 177microns. Subject to size reduction the size will differ by the methodused and the source of the product. A smaller physical size is alsopossible. Smaller sizes may be more soluble.

In addition, PCA crystals can be subject to physical grinding or jetspraying to make into a powder. The smaller size is less than that of ahuman red blood cell. The rbc has a disk diameter of approximately6.2-8.2 μm and a thickness at the thickest point of 2-2.5 μm and aminimum thickness in the center of 0.8-1 μm. This post ground size ismuch smaller than most human cells.

Co-crystals of PCA is another means to coat skin or hard surfaces. Thesolubility of PCA in water is approximately 10 mg/ml and LD50 is 800mg/kg which makes PCA a potential co-crystal former. Seven novelcocrystals of PCA with ε-Caprolactam, Isonicotinamide, Isonicotinic acid(hydrated and anhydrate cocrystals), Theophylline, Nicotinamide andCarbamazepine are known.

One of the major proteins of COVID 19 is Mpro (main protease), alsoreferred to as the “3C-like protease” belonging to the proteases classof hydrolytic enzymes. This enzyme plays a key role in the processing ofpp1a (responsible for generating copies of viral genome) and pp1ab(responsible for generating viral genome) as involved in theirproteolytic cleavage at the conserved residues among COVID 19 genome.

These can assemble to give rise to virions inside a host cell and thus,replicate to produce multiple copies. Mpro can act as potential targetfor structure-based drug discovery as this enzyme not only involved inautocatalytic cleavage of itself and key viral enzymes, as well as lacksany close homologues among human host. Targeting this enzyme usingsuitable protease small molecule inhibitor can curb virus replicationand transcription which are critical steps in virus life cycle.

Molecular docking is an important tool in computer-based drug design anddrug discovery which helps to predict the small ligand conformation andorientation (Docking pose) within the active sites of the targetreceptor protein.

Protocatechuic acid has high docking score (−9.8) and importantlyprotocatechuic acid derivatives show comparatively betterpharmacokinetic predictions and lead likeness, along with the ease ofsynthesis.

PCA as one of the polyphenolic scaffolds have affinity to bind withsubstrate-binding pocket of COVID-19 virus Mpro, which is highlyconserved among all CoV Mpros. This shows that small molecule inhibitors(PCA) targeting Mpro or in combination with other adjuvant therapies mayprovide an effective therapeutic regime to fight against all coronavirusassociated diseases.

In embodiments, crystals of PCA can thus be placed in a liquid vehiclefor delivery to a target, for example, skin or personal protectivecoating. Accordingly, in embodiments, the present disclosure providescoating of various products including personal protective equipment andhard surfaces. Smaller sized crystals may also be used for intravenousand aerosol delivery to a patient.

Oral and Nasal applications of PCA crystals may use water as a vehicleand, in embodiments, at a maximal concentration of about 1.24% PCA. Thevehicle may be sterile water and or saline solution with a minimalamount of an essential oil or other for stability. PCA may be combinedwith a cellulose vehicle in nasal topical application. As mentioned,safety is inherent as PCA is FDA approved as a food flavoring additiveas are the essential oils. Therefore, any entry into or beyond the oralcavity is inherently safe.

In embodiments, personal protective equipment may be coated or infusedwith PCA crystals. Coatings may be applied at the time of manufacture orafter-market, i.e., while in use. In embodiments, an alcohol-basedsolution with 1 to 30% PCA may be used depending upon the intendedapplication. The typical amount might be about 3% in a spray container,apparatus and/or mechanized delivery at manufacture of the article aswith robotics and or assembly line.

In this embodiment, the alcohol vehicle causes an immediate 99% plusviral kill when wet. Upon drying, PCA crystals remain as an anti-viralcoating. PCA in this form on metal and or cloth destroys SARS CoV-2virus on contact. The anti-viral function can be based upon the PCAcrystals in solution, and or dry, and cause the SARS CoV-2 virus to bedead on arrival (D.O.A.) due to subsequent contact.

In embodiments, benefits for protective masks is clear since studieshave shown masks to harbor the SARS-Co-2 virus for up to 6 days. Facemasks have limitations since there must be air flow for breathing. TheN95 mask is promoted as the best alternative. However, those with avalve will allow particles to escape. Thereby they may protect thewearer, but if the wearer is contagious there is no protection forothers. The 3M mask known as the 8511 Respirator with a Cool Flow Valvemade by 3M is made for construction workers to control dust and allowfor easy breathing.

The particle size of the SARS CoV-2 virus is relatively large for avirus but is less than 0.1 microns or 125 nanometers. It is noted thateach particulate respirator is given a filter efficiency rating of 95,99, or 100 when tested against particles that are the most difficultsize to filter—approximately 0.3 microns in size mass median aerodynamicdiameter (MMAD). NIOSH class 95 particulate respirator filters arecertified to be at least 95% efficient. It is noted that 100% is notpossible. Also, it is noted that the virus is 0.1 micron and the openingin this mask is 0.3 micron.

The present disclosure thus provides important additional protection forfiltering. This is accomplished with a coating on the filter so that anyvirus passing through will be destroyed, and much more so than if nocoating of PCA is present.

The disclosure further provides for spraying or wiping of surfacesincluding hard surfaces with PCA in an alcohol vehicle which results inimmediate anti-viral removal. In addition, the anti-viral crystalcoating on hard surfaces like handles, doorknobs, gas pumps will thenremain and continue to kill virus for a significant period afterwards.

The supporting experimental tests described below replicate a clinicalapplication in several ways. First, is the effectiveness of the depositof dry crystals on hard surfaces; metal and plastic. Then theapplication to apply dry crystals to N95 outer mask material. Unique tothe testing methods was that the virus was delivered in a liquid medium;alcohol, propylene glycol, essential oil and/or water. Therefore, thetests on virus assessed the effect of crystals of PCA in a solution toinactivate the SARS Co-2 virus in a liquid medium. This replicates theclinical transmission of the SARS Co-2 virus which is aerophilizeddroplets means of spreading in a liquid medium, and the virus isinactivated by the PCA crystals in solution.

As used herein, the term virucidal generally means having the capacityto or tending to destroy or inactivate viruses.

The term facility generally refers to any public facility. Businesses,hospitals, gas stations, restaurants, public rest stops, including restrooms, etc., airports, bus stations, stadiums and public entertainmentfacilities, public transportation stations and vehicles, movie theaters,health care facilities, medical offices, clinics, outpatient surgerycenters, and any other business or facility where the public gathersand/or transits.

The term personal protective equipment includes face coverings andmasks, gowns, aprons, gloves, shoes and shoe coverings, and any otherarticle used for personal protection particularly used to protect fromthe transmission of viruses and bacteria.

The term ‘room’ generally means an enclosed space in a buildingparticularly public facilities and buildings, hospitals, publictransportation facilities, restaurants, entertainment facility, reststops, or any other enclosed space where the public may gather.

A stabilizer may be a chemical that is used to prevent separation ordegradation. Stabilizers can include emulsifiers and surfactants, forexample, for stabilization of emulsions.

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a composition” includesmixtures or combinations of two or more such compositions.

Throughout the specification and claims, the word “comprise” andvariations of the word, such as “comprising” and “comprises,” means“including but not limited to,” and is not intended to exclude, forexample, other components, extracts, additives, or steps. It is alsocontemplated that embodiments described as “comprising” components, theinvention also includes those same inventions as embodiments “consistingof” or “consisting essentially of.”

Ranges can be expressed herein as “approximately” or from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes from the oneparticular value and/or to the other particular value.

A weight percent of a reagent, component, or compound unlessspecifically stated to the contrary, is based on the total weight of thereagent, component, composition or formulation in which the reagent,component, or compound is included, according to its usual definition.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant decrease or lower a characteristic (e.g.,inflammation, growth, or viability of viruses).

By “promote” or other forms of the word, such as “promoting,” is meantto induce a particular event or characteristic, or delay the developmentor progression of a particular event or characteristic, or to minimizethe chances that a particular event or characteristic will occur.

“Treat” or other forms of the word, such as “treating,” “treatment” ortreated,” is used here to mean to administer a composition or to performa method in order to induce, reduce, eliminate, and prevent acharacteristic (e.g., inflammation, growth or viability of viruses). Itis generally understood that treating involves providing an effectiveamount of the composition to the mammal or surface for treatment.

The term “vehicle” or “vehicle carrier” as used herein refers to meanthe manner in which the reagents or compositions may be delivered,including as a liquid, salve, soap, foam, cream, solution, gel, spray,powder, wipes, antiviral treatments, wipes, and the like.

The term fog can generally mean a cloudlike mass or layer of minutedroplets including crystals. The term fogging can mean to cover orenvelop with a fog. Fogging can be accomplished with fogging ordisinfecting machines well known in the art. For example, medical gradedisinfecting fogging machines can preferably be used. Portable medicalgrade fogging machines are also preferred. Large scale industrialfogging machines can be used for large facilities like hospitals,stadiums, public entertainment facilities, etc.

A fog machine, fog generator, or smoke machine is generally a devicethat emits a dense vapor that appears similar to fog or smoke. Anartificial fog may be used in large scale applications, and smaller,more affordable fog machines can be used for more localizedapplications. Fog machines typically used in a variety of industrial,training, health care, and military applications are contemplated.Typically, fog is created by vaporizing proprietary water andglycol-based or glycerin-based fluids (or through the atomization ofmineral oil). This fluid (often referred to colloquially as fog juice)vaporizes or atomizes inside the fog machine. Upon exiting the fogmachine and mixing with cooler outside air the vapor condenses,resulting in a thick visible fog.

Spraying is generally a fluid flying in small drops or particles forexample mechanically created and blown and may be a jet of vapor orfinely divided liquid and can be produced by a device (such as anatomizer or sprayer) by which the spray is created, dispersed, and/orapplied.

By “additive” or “food additive” is meant to the use as a component ofany food (including any substance intended to use in producingmanufacturing, packing, processing, preparing, treating, packaging,transporting, or holding food).

By “antiseptic” is meant an antimicrobial reagent or composition that isapplied to any surface, including skin or tissue, to effect (e.g.,eliminate, inhibit, decrease or prevent) viral growth, viability, and/orsurvival.

By “disinfect” or other forms of the word, such as “disinfectant” or“disinfecting,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) viral growth,viability or survival at any concentration. It is generally understoodthat disinfect involves providing an effective amount of the compositionto any surface, but particularly solid surfaces, whether smooth orporous or semi-porous, or cloth-like surfaces.

By “sanitize” or other forms of the word, such as “sanitizer” or“sanitizing,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) viral growth,viability or survival at any concentration. It is generally understoodthat sanitizing involves providing an effective amount of thecomposition to any surface. Further, it is generally understood thatsanitizing solutions and sanitizing components are those solutions thatmay be safely used on food-processing equipment and utensils and onother food-contacting conditions.

By “sterilize” it is meant to kill on the article being sterilized.Sterilize and sterilization include cold sterilization methods.

By “isolated” or “an isolate” as it refers to either the compounds orreagents described herein means not 100% by weight but ratherapproximately 95% to 97% of the compound or reagent by weight.

The term “alkyl” as used herein is a branched or unbranched hydrocarbongroup of 1 to 20 carbon atoms. Non limiting examples include methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl,eicosyl, tetracosyl, and the like. Further, the alkyl group can also besubstituted or unsubstituted.

The term “alkoxy” or “alkyoxy group” as used herein refers to a branchedor unbranched hydrocarbon chain having from 1 to 15 carbons and linkedto oxygens. Non-limiting examples include methoxy, ethoxy and the like.

The term “medicament” as used herein refers to any wound treatment,including but not limited to the group consisting of burn reliefmedications, anesthetic agents, wound cleansers, antiseptic agents, scarreducing agents, immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,acne treating agents, sunscreen agents, dermatological agents,antihistamine agents, antibacterial agents, bioadhesive agents,inhibitors of prostaglandin synthesis, antioxidants, and mixturesthereof.

Unless stated to the contrary, a formula contemplates each possibleisomer.

The term “nutraceutical” as used herein refers to any food stuff,including a dietary supplement or fortified food, provided for potentialhealth and medical benefits.

The present invention also provides methods, compositions and uses fortreating surfaces (solid, smooth, porous or semi-porous, or cloth-like)and liquids to reduce viral growth or to sanitize or sterilize thesurface. More specifically, the methods and compositions describedherein include contacting any surface with a composition comprising PCAthereby reducing or preventing viral growth on said surface, or tosanitize or sterilize the surface. The surfaces can be in the healthcare setting, sports setting, including stadiums, or even foodpreparation settings or any setting where sterile surfaces are required.

The compositions can be applied to solid surfaces such as implants, orsolid surfaces like operating tables, benches, equipment, patient beds,etc. or surgical instruments to sanitize or sterilize the surface Solidsurfaces such as operating tables, other equipment and other surfacescan be treated as well by spraying of the surface with compositionscomprising PCA.

In addition, the compositions can be applied to smooth, porous orsemi-porous, or cloth-like surfaces such as wound dressings, bedding,vascular implants, bandages, etc. The material can be treated with thePCA solution and then used immediately, or the material can be allowedto dry and then used. For example, a bandage can be treated with PCA andthen allowed to dry and store (for about up to 2 years known shelflife). When needed, depending upon the nature of the wound, the bandagecan either be applied directly to the wound or can be wetted with water,70-90% isopropyl alcohol, saline or propylene glycol and/or essentialoils and then applied to the wound. It is preferred to use propyleneglycol and an essential oil as they enhance the absorption of PCA intothe skin.

When the composition of this invention is applied to as an element of acovering or bandage, to adhere to a surface to be treated, such as awound, the composition generally can include a concentration of the PCAof at least 1.24% and 30% by weight of the compound depending upon thechemical nature of the vehicle and the target. In certain embodiments a25 mM concentration of PCA is applied to a bandage.

Further, the compounds of this invention will be between 20-30% byweight of the compound for one intended use and more preferably, between1.24% and 30% by weight of the compound depending upon the chemicalnature of the vehicle, the target being treated and the species of virusto be treated.

Further, the methods and compositions described herein include addingthe composition comprising PCA thereof to liquid or fluid, includingother sanitizing solutions and/or sanitizing components. Further still,the methods and compositions described herein include adding thecomposition comprising PCA to any other vehicle, including but notlimited to a powder, paste, cream foam, gel, wipes, other sanitizingcomponents and the like thereby killing, reducing or preventing viruseson said surface.

The present invention provides a composition that destroys virusesincluding the Covid-19 virus. The composition includes PCA. The PCA maybe mixed with 70% isopropyl alcohol and or small amount of essentialoil, i.e. lemon, peppermint, etc. The concentration of PCA can beanywhere from about 20% by weight PCA to 100% PCA. Preferably theconcentration of PCA varies with the intended purpose from 1.24%, 20%,30%, 20-50% or 20-40% or 20-30% by weight. When the PCA is used in acrystal form, then the crystals can be up to 100% PCA by weight. The PCAmay be in the form of crystals that are embedded into a material such asa cloth or a mesh, such as titanium or stainless steel. The crystals maybe applied to a surface configuration that provides for housing of thecrystal on the surface. The same is for cloth material that has mesh orsurface to house the physical crystals by size. They may remain in placein crystal form until activated, for example, when subject to fluidcommon to the mammalian body.

The amount of PCA necessary for coating metal and or cloth may be 20 to30% PCA or 20-30 grams per 100 ml of 70% isopropyl alcohol. Thesecompositions allow for higher concentrations, and evaporate rapidly todry state of PCA crystals on the surface or cloth.

In another aspect of the present invention, a method of disinfecting asurface comprising contacting said surface with PCA is contemplated.

This disclosure also provides for a method comprising contacting asurface with an effective amount of the composition. By the term“effective amount” of a composition as provided herein is meant anamount of a composition sufficient to provide the desired benefit. Withany prescription; onset of treatment, dose, vehicle, route, interval,frequency or duration the fundamental therapeutic action is theengagement of one or more crystals with the SARS CoV2 virus in a liquidenvironment; in or out of the mammalian body. As disclosed herein, theexact amount required will vary from use to use depending on a varietyof processing parameters, as understood by one of ordinary skill, suchas the application, type of surface, the type of virus to be treated,the surface size, the mode of delivery (e.g., aerosol, spraying ordipping), and the like. Determination of what constitutes an “effectiveamount” is made by routine testing with known concentrations andadjusting those concentrations as needed to obtain the desired benefitand can be determined by one of ordinary skill in the art using routineexperimentation so that the PCA crystals engage the SARS CoV-2 virus.

When the antiviral composition of this invention is applied to a surfaceto be treated, the antiviral composition generally can include aconcentration of the PCA, not including the carrier. Further, the PCAcan be between 90%-97% by weight of the compound, and more preferably,between 95%-98% by weight of the compound.

When the antiviral composition or compositions of this invention areapplied to a surface to be treated may be diluted for use as a sanitizeror as a preventive or prophylactically, and at greater concentrationsfor treatment.

In another aspect of the invention provides a method of inhibitinggrowth of a virus on a solid, smooth, porous or semi-porous, orcloth-like surface (such as but not limited to a cloth, wound dressing,bandage, heart or vessel grafts) by treating the surface with acomposition of the present invention, preferably comprising PCA. Thesurface can be any solid, smooth, porous, or semi-porous, or cloth-likesurface.

The present invention thus provides a composition that destroys orinhibits a virus. The composition is preferably PCA. The PCA may bemixed with 70% isopropyl alcohol. The concentration of PCA can beanywhere from about 20% PCA to 100% PCA. Preferably the concentration ofPCA is about 20-50% by weight, or is about 20-40% by weight or is about20-30% by weight or is 30% to 50% by weight. Additionally, the PCA maybe in the form of crystals that are embedded into a material such as acloth or a mesh, such as titanium or stainless steel.

The invention provides another composition comprising about 17 to 40%,or 17 to 30% or 17 to 20% by weight of PCA, isopropyl alcohol, propyleneglycol and an essential oil, preferably of peppermint, or a citrus fruit(i.e. lemon, grapefruit, orange, lime, etc.). This composition is usefulin the methods described in the invention, for example as a skinantiseptic as a surface disinfectant, as a spray to disinfect a surface,etc. The composition of the invention, may have at least PCA at 17+% byweight in at least 70-90% isopropyl alcohol, propylene glycol (15 mls ina 105 ml total solution) and the essential oil; i.e. peppermint or lemonetc.

The invention further provides a composition of PCA wherein thecomposition comprises or consists of PCA that can be applied directly orprovided in various vehicles depending upon the application. Acomposition of 70% isopropyl alcohol, propylene glycol and essentialpeppermint oil may be effective in use as a skin antiseptic. Higherconcentration of 10% PCA (20 grams in 90 milliliters of 70% isopropylalcohol) may be more effective than PCA in water. The followingconcentration may also be effective—the composition comprising orconsisting of PCA (20 grams) 70% isopropyl alcohol (85 ML), propyleneglycol (15 ml) and an essential oil (5 ml).

In addition to the components and administration of said compositionsdisclosed above, the compositions can be in the form of an aqueoussolution and also deliver PCA in suspension and or upon drying produce aresidual PCA coating. The compositions disclosed herein can also be inthe form of a liquid, gel, suspension, dispersion, solid, emulsion,aerosol, for example, powders, tablets, capsules, pills, liquids,suspensions, dispersions or emulsions. Also, the compositions disclosedherein can be in the form suitable for dilutions. Similarly, thecompositions can be in the form of a powder, cream, paste, gel or solidthat can be reconstituted.

Other components can be present in the composition, if desired. Forexample, the antiviral composition can also include at least oneadditive selected independently from a carrier, a diluent, an adjuvant,a solubilizing agent, a suspending agent, a filler, a surfactant, anantimicrobial agent, a preservative, a viscosity modifier, a thixotropymodifier, a wetting agent, an emulsifier, or any combinations thereof.For example, the disclosed antiviral composition can further comprise atleast one surfactant selected from a cationic surfactant, an anionicsurfactant, a non-ionic surfactant, and an amphoteric surfactant.Additionally, the disclosed antiviral and/or pharmaceutical compositionsmay further comprise medicament is selected from the group consisting ofburn relief medications, anesthetic agents, wound cleansers, antisepticagents, scar reducing agents, immunostimulating agents, anti-bacterialagents, biofilm destroying agents, antiviral agents, antikeratolyticagents, anti-inflammatory agents, antifungal agents, acne treatingagents, sunscreen agents, dermatological agents, antihistamine agents,antibacterial agents, bioadhesive agents, inhibitors of prostaglandinsynthesis, antioxidants, and mixtures thereof.

Also, the disclosed antiviral compositions can optionally include one ormore additives such as carriers, adjuvants, solubilizing agents,suspending agents, diluents, surfactants, other antiviral orantimicrobial agents, preservatives, fillers, wetting agents,antifoaming agents, emulsifiers, and additives designed to affect theviscosity or ability of the composition to adhere to and/or penetrate awound.

In one embodiment, the disclosed antiviral compositions, including theselected active components, including PCA are without causingsignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained.

In another embodiment, antiviral compositions of the present inventionare formulated for use in liquids, solutions, gels, soaps, creams,powders, salves, and other preparations designed for topical use asantiseptic agents, sprays, foams, antibacterial treatments, wipes andthe like. In another embodiment, antiseptic compositions of the presentinvention are formulated as a hand antiseptic, sanitizer ordisinfectant.

In yet another embodiment, sanitizing compositions of the presentinvention are formulated for use in liquids, solutions, gels, soaps, andother preparations designed for use as sanitizing agents, liquids,including sprays, foams, gels, soaps, sanitizing treatments, and thelike when used as a sanitizing solution, including but not limited to,use in food processing facilities, including food-processing equipmentand utensils, and on other food-contact articles.

In yet another embodiment, sanitizing compositions of the presentinvention use in food processing facilities, including food-processingequipment and utensils, and on other food-contact articles areformulated to include any components generally recognized as safe foruse in food processing facilities, including but not limited to, aqueoussolutions containing potassium, sodium or calcium hypochlorite, asolution of hydrogen peroxide, an aqueous solution containing potassiumiodide, sodium lauryl sulfate, sodium-toluenesulfonchloroamide,solutions containing dodecylbenzensulfonic acid, other acceptabledetergents and the like.

In another aspect, the compositions of the present invention are used infood processing, packing, manufacturing, handling, preparing, treating,transporting, or holding as a food additive without causing undesirableeffects or interacting in a deleterious manner. By way of example,protocatechuic acid can be used as an additive in meat, including thehandling and processing, without causing undesirable effects orinteracting in a deleterious manner with the meat.

In yet another aspect, the compositions of the present invention areused in food processing, including cold sterilization of foodcontainers, including bottles, without causing undesirable effects orinteracting in a deleterious manner.

In examples, the antiviral compositions disclosed herein can furthercomprise a carrier or vehicle. The term “carrier or vehicle” means acompound, composition, substance, or structure that, when in combinationwith a compound or composition disclosed herein, facilitatespreparation, administration, delivery, effectiveness, or any otherfeature of the compound or composition. Examples of carriers includewater, isopropyl alcohol ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), vegetable oils, andsuitable mixtures thereof. “Pharmaceutically acceptable carrier” means acompound, composition, substance, or structure that is useful in neitherpreparing a pharmaceutical composition which is generally safe,non-toxic, and neither biologically nor otherwise undesirable andincludes that which is acceptable for veterinary as well as humanpharmaceutical use.

In a further example, the antiviral compositions disclosed herein canalso comprise adjuvants such as preserving, wetting, emulsifying,suspending agents, and dispensing agents.

Suitable suspending agents can include, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, or mixtures of these substances, and the like.

The disclosed antiviral compositions can also comprise solubilizingagents and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl alcohol,benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,oils, in particular, cottonseed oil, groundnut oil, corn germ oil, oliveoil, castor oil and sesame oil, glycerol, tetrahydrofur fury 1 alcohol,polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like. The additives can be present in thedisclosed compositions in any amount with the PCA.

Example 1

In one example PCA was deposited on the skin. It was staged at a higherconcentration for illustrative purposes. The PCA forms a layer andprotective barrier on the skin.

Example 2

In a second example, PCA crystals were applied to a metallic surface onemonth prior. A 2×0.5 mm bare area in the middle could not be wiped off,and the crystals were scraped off with a fingernail and some resistance.A pile up of crystals occurred on a right end of the scrape.

Example 3

In a third example a steel utensil was used in stirring a 30%concentration of PCA in ethanol. A PCA coating formed which was visible,uniform and quickly applied.

Example 4

Example 4 demonstrates the results of two independent contractlaboratory studies utilizing a methodology replicating the clinicaltherapeutic environment whereby the PCA crystal engages the SARS CoV-2virus in an aqueous environment. These studies demonstrate theeffectiveness of Protocatechuic Acid (PCA) against SARS-CoV-2, thecausative virus for COVID19.

The Test Article (TA) used for this study was Protocatechuic Acid (PCA).The TA was received as an off-white powder. The PCA solution wasprepared to be 30% PCA w/v in Ethanol. The PCA was prepared in 5 gincrements to pre-warmed 50-60 mL ethanol until dissolved for a total of30 g PCA in the solution. Additional ethanol was then addedvolumetrically to be equivalent to 100 mL.

The Test Substrates (TS) were a Plastic-type material sourced from aclear plastic laboratory bottle (Corning 431731 Octagonal bottle, 150mL), cloth (the top layer of a N95 mask [3M 8210]), and aSponsor-provided wire mesh to serve as a substrate for the TA. All testsubstrates were cut to approximately 1″×1″ in size. The test substrateswere submerged into the PCA solution and dried horizontally to allow foreven coating. After the substrate was thoroughly dried, the testsubstrate was re-submerged into the PCA solution for an additionalcoating.

The Test Virus used for this study was 2019 Novel Coronavirus, IsolateUSA-WA1/2020 (SARS-CoV-2). The virus was stored at approximately −65° C.prior to use. The multiplicity of infection (MOI) was 0.01 TCID₅₀/cell.

The Cell Culture used for the TCID₅₀ test was African Green MonkeyKidney Cells (Vero E6 cells) that were maintained in Dulbecco's MinimumEssential Medium with 10% fetal calf serum (DMEM-2). All growth mediacontained heat-inactivated fetal calf serum and antibiotics.

The test design is shown below in Table 1. This test assesses the TA ona substrate in various conditions as shown in Table 1.

The Test Substrate was coated with PCA as described above. The testsubstrates was treated with PCA twice and allowed to fully dryovernight. In general, the time from the first coat to the next day'svirus exposure was approximately 24 hours.

The treated Test Substrate plus TA was placed into a sterile 6 well cellculture plate and approximately 100 μL total of a ≥1×10⁶ TCID₅₀/mLSARS-CoV-2 virus was such that 50 μL of virus was layered on each sidesof the treated test substrates. This was the procedure used for theinitial Day 1 experiment.

For the confirmatory test, in an attempt to increase the recorded titerof the controls, the treated Test Substrate plus TA was placed into asterile 6 well cell culture plate and the same amount of virus waslayered onto both sides of the test substrate. However, an addition 50μL of DMEM was added to each side to reduce the inactivation of thevirus due to desiccation. Additionally, a glass coverslip was also addedto help mitigate against evaporation.

After application of the virus, the virus was contact with the Testsubstrates for approximately 10 minutes (Groups 1, 2, and 3, Controlgroups 7, 8, and 9), 60 minutes (Groups 4, 5, and 6, Control Groups 10,11, and 12). Each substrate per time per test article was performed induplicate.

A cell culture-only control was included to indicate that cells withoutany TA or virus remain healthy throughout the assay. Virus-only controlswithout substrate was added for each timepoint to verify that the assaywas performing as expected.

After the incubation time, the treated substrate was washed with 1 mL ofcell culture media (DMEM-2) for approximately 5-10 minutes within the 6well cell culture plate and the glass cover slip removed if necessary.This was the equivalent to a 10-fold dilution. The plate was gentlystirred via an orbital shaker to enhance the recovery of the virus.

For the TCID₅₀, the cell culture media (DMEM-2) used to wash the TestSubstrate was serially diluted 10 fold and transferred into respectivewells of a 96-well plate which contained a monolayer of African GreenMonkey Kidney Cells (Vero E6 cells) for titration. The TCID₅₀ assay wasperformed non-GLP according to IITRI Standard Operating Procedures forthe assay. The TCID₅₀ titers was calculated using the method ofReed-Meunch.

TABLE 1 Study Design Group Test and Control Groups PCA 1 Plastic (10minute exposure) 2 replicates 2 Cloth (10 minute exposure) 2 replicates3 Mesh (10 minute exposure) 2 replicates 4 Plastic (60 minute exposure)2 replicates 5 Cloth (60 minute exposure) 2 replicates 6 Mesh (60 minuteexposure) 2 replicates 7 Virus Control-Plastic (10 minute 2 replicatesexposure) 8 Virus Control-Cloth (10 minute exposure) 2 replicates 9Virus Control-Mesh (10 minute exposure) 2 replicates 10 VirusControl-Plastic (60 minute 2 replicates exposure) 11 Virus Control Cloth(60 minute exposure) 2 replicates 12 Virus Control Mesh (60 minuteexposure) 2 replicates

The Test Articles, Test Substrates and virus (SARS-CoV-2) were preparedaccording to protocol and each preparation was noted in the studynotebook for this study.

Two experimental days were run for this study with the second day as runas a confirmatory. For Day 1, after coating the Test Substrates with PCAas described above (Groups shown in Table 1 above), a TCID₅₀ wasperformed at 10 minutes or 60 minutes after initial application of thevirus. There was an observed log difference between the experimentalgroups (Group 1: Plastic-10 min, Group 2: Cloth-10 min, Group 3: Mesh-10min, Group 7: Plastic-60 min, Group 8: Cloth-60 min, Group 9: Mesh-60min) when compared to the controls (Group 4: Plastic-10 min, Group 5:Cloth-10 min, Group 6: Mesh-10 min, Group 10:Plastic-60 min, Group 11:Cloth-60 min, Group 12: Mesh-60 min)

Day 1 results observed did indicate some log reductions in infectiousvirus titers under the experimental conditions performed for this studywhen compared to controls. The results are shown below in Table 2.

TABLE 2 Initial Experimental Run Results. Test Incubation TCID₅₀ Log logGroup Article/substrate Replicate time Log₁₀/mL* average St. Dev.difference{circumflex over ( )} 1 PCA/plastic 1 10 Min 3.75 3.75 0.00−0.63 PCA/plastic 2 10 min 3.75 2 PCA/Cloth 1 10 min 2.75 2.75 0.00−1.25 PCA/Cloth 2 10 min 2.75 3 PCA/Mesh 1 10 min 3.50 3.38 0.18 −0.25PCA/Mesh 2 10 min 3.25 4 Control/plastic 1 10 Min 3.75 4.38 0.88 N/AControl/plastic 2 10 min 5.00 5 Control/Cloth 1 10 min 3.75 4.00 0.35N/A Control/Cloth 2 10 min 4.25 6 Control/Mesh 1 10 min 3.75 3.63 0.18N/A Control/Mesh 2 10 min 3.50 7 PCA/plastic 1 60 Min 3.25 2.88 0.53−1.13 PCA/plastic 2 60 Min 2.50 8 PCA/Cloth 1 60 Min 2.50 2.75 0.35−1.00 PCA/Cloth 2 60 Min 3.00 9 PCA/Mesh 1 60 Min 1.00 1.50 0.71 −2.00PCA/Mesh 2 60 Min 2.00 10 Control/plastic 1 60 Min 3.75 4.00 0.35 N/AControl/plastic 2 60 Min 4.25 11 Control/Cloth 1 60 Min 4.00 3.75 0.35N/A Control/Cloth 2 60 Min 3.50 12 Control/Mesh 1 60 Min 3.25 3.50 0.35N/A Control/Mesh 2 60 Min 3.75 13 Virus control N/A 10 min 5.75 N/A N/AN/A (no coupon) 14 Virus control N/A 60 min 5.75 N/A N/A N/A (no coupon)*limit of detection is 1.5 TCID₅₀ Log₁₀/mL {circumflex over ( )}Logdifference is defined as the averaged TCID₅₀ Log₁₀/mL from virus controlon substrates - TCID₅₀ Log₁₀/mL from replicate test group. Logdifference indicates amount of reduction in infectious virus whencomparing the virus control on substrate to the test group.

For Day 2, after coating the Test Substrates with PCA as described above(Groups shown in Table 1 above), a TCID₅₀ was performed at 10 minutes or60 minutes after initial application of the virus. There was amodification to the procedures to see if the viral titers could beincreased. To mitigate against evaporation during the incubationperiods, these modifications included adding an additional 50 μl of DMEMon each side of the test substrate and a glass coverslip was placed ontop of the test substrate. As with the Day 1 run, there was an observedlog difference between the experimental groups (Group 1: Plastic-10 min,Group 2: Cloth-10 min, Group 3: Mesh-10 min, Group 7:Plastic-60 min,Group 8: Cloth-60 min, Group 9: Mesh-60 min) when compared to thecontrols (Group 4:Plastic-10 min, Group 5: Cloth-10 min, Group 6:Mesh-10 min, Group 10:Plastic-60 min, Group 11: Cloth-60 min, Group 12:Mesh-60 min) as shown in Table 3, thereby confirming the results fromthe Day 1 run.

TABLE 3 Confirmatory Experimental Run Results. Test Incubation TCID₅₀Log log Group Article/substrate Replicate time Log₁₀/mL* average St.Dev. difference{circumflex over ( )} 1 PCA/plastic 1 10 Min 4.25 4.380.18 −1.13 PCA/plastic 2 10 min 4.50 2 PCA/Cloth 1 10 min 4.25 4.25 0.00−1.13 PCA/Cloth 2 10 min 4.25 3 RCA/Mesh 1 10 min 4.75 4.63 0.18 −1.13RCA/Mesh 2 10 min 4.50 4 Control/plastic 1 10 Min 5.50 5.50 0.00 N/AControl/plastic 2 10 min 5.50 5 Control/Cloth 1 10 min 5.50 5.38 0.18N/A Control/Cloth 2 10 min 5.25 6 Control/Mesh 1 10 min 5.75 5.75 0.00N/A Control/Mesh 2 10 min 5.75 7 PCA/plastic 1 60 Min 3.50 3.63 0.18−1.50 PCA/plastic 2 60 Min 3.75 8 PCA/Cloth 1 60 Min 2.00 2.75 1.06−2.38 PCA/Cloth 2 60 Min 3.50 9 RCA/Mesh 1 60 Min 4.50 4.38 0.18 −0.88RCA/Mesh 2 60 Min 4.25 10 Control/plastic 1 60 Min 5.00 5.13 0.18 N/AControl/plastic 2 60 Min 5.25 11 Control/Cloth 1 60 Min 4.50 5.13 0.88N/A Control/Cloth 2 60 Min 5.75 12 Control/Mesh 1 60 Min 5.25 5.25 0.00N/A Control/Mesh 2 60 Min 5.25 13 Virus control (no N/A 10 min 5.75 N/AN/A N/A coupon) 14 Virus control (no N/A 60 min 5.75 N/A N/A N/A coupon)*limit of detection is 1.5 TCID₅₀ Log₁₀/mL {circumflex over ( )}Logdifference is defined as the averaged TCID₅₀ Log₁₀/mL from virus controlon substrates - TCID₅₀ Log₁₀/mL from replicate test group. Logdifference indicates amount of reduction in infectious virus whencomparing the virus control on substrate to the test group.

TABLE 4 Comparison between Initial Experimental Run to Confirmatory RunTest Article/ Incubation Day 1: Log Confirmatory: substrate timedifference Log difference PCA/plastic 10 Min −0.63 −1.13 PCA/Cloth 10min −1.25 −1.13 PCA/Mesh 10 min −0.25 −1.13 PCA/plastic 60 Min −1.13−1.50 PCA/Cloth 60 min −1.00 −2.38 PCA/Mesh 60 min −2.00 −0.88

A PCA coating on the three test substrates, appeared to show someeffectiveness in reducing infectious virus titers in the experimentalcondition shown in the protocol after the 10 minutes and 60 minutespost-exposure incubation when compared to the virus control onsubstrate. From both the Day 1 and the confirmatory runs, the logreduction varied between a 0.63 to a 2.38 log reduction.

Overall, these results show that PCA when coated approximately 24 hoursprior to virus exposure can reduce infectious virus performance on asubstrate, however, overall effectiveness was somewhat varied betweenruns and test substrate. Additionally, it appears that a longerincubation time may be marginally more effective than the shorter 10minute time. A 1 to 2 log reduction/difference corresponds to a 90 to99% inactivation while a 3 log reduction corresponds to a 99.9%inactivation.

Example 4

The second laboratory utilized test coupons made of solid stainlesssteel, plastic and K95 mask were coated in 30% w/v PCA in 70% ethanol.Each coupon was dipped in PCA, allowed to dry, dipped again and allowedto dry with the opposite side of the coupon facing up. Once dry, 200 ulvirus was added to each coupon and allowed to dry (45 minutes-1 h dryingtime). Virus was recovered by adding 2 ml DMEM/F12 media and washing thecoupon, without scraping so as not to dislodge PCA crystals. A yellowcolor change in the media was observed indicating acidification of themedia upon addition to the PA-coated coupon. The recovered virus wasadded to empty 96 well plates and diluted 1:10 down the plate. This wasthen added to Vero E6 cells that had grown to ˜70% confluence.Cytotoxicity controls without virus and recovery controls without PCAwere also done in the same manner. After addition to the cells, plateswere read at day 4 for the presence of cytopathic effect (CPE) due toviral infection of cells. Note that cytotoxicity and CPE cannot bedifferentiated in this assay, thus any dead cells are marked aspositive.

Cytotoxicity was seen up to 1:100 dilution for the K95, and 1:10 for thestainless steel and plastic coupons. Positive CPE for virus recoverycontrols was seen at least down to 1:10,000 dilutions for all 3 couponmaterials, thus each coupon material was adequate for coupon testing.Results are shown in the table below. The SS=stainless steel, K95=K95mask and P=plastic. +PCA means coupons coated with PCA. No PCA (e.g.SS-1) indicates virus recovery controls with no PCA coating that hadvirus dried and recovered.

TABLE 5 Log Average Reduction Percent Sample Log10 Average Log10 toVirus Log Name Replicate# TCID50 TCID50/mL TCID50 TCID50 TCID50 ControlsReduction SS + PCA-1 1 501.1872 0.01995262 2.70 298.9493 2.37 2.9399.88% SS + PCA-2 2 79.43282 0.12589254 1.90 SS + PCA-3 3 316.22780.03162278 2.50 SS-1 1 87992.25 0.00011365 4.94 226075.8 5.29 SS-2 2316227.8 3.1623E−05 5.50 SS-3 3 274007.4 3.6495E−05 5.44 K95 + PCA-1 1316.2278 0.03162278 2.50 182.4589 2.10 2.45 99.65% K95 + PCA-2 2199.5262 0.05011872 2.30 K95 + PCA-3 3 31.62278 0.31622777 1.50 K95-1 158230.63 0.00017173 4.77 39285.11 4.55 K95-2 2 19952.62 0.00050119 4.30K95-3 3 39672.07 0.00025207 4.60 P + PCA-1 1 50.11872 0.19952623 1.7088.00117 1.91 3.94 99.99% P + PCA-2 2 125.8925 0.07943282 2.10 P + PCA-33 87.99225 0.11364637 1.94 P-1 1 1217075 8.2164E−06 6.09 971841.2 5.86P-2 2 203950 4.9032E−05 5.31 P-3 3 1494498 6.6912E−06 6.17

While this disclosure includes specific examples, it will be apparentafter an understanding of the disclosure of this application has beenattained that various changes in form and details may be made in theseexamples without departing from the spirit and scope of the claims andtheir equivalents.

The claimed invention is:
 1. A nasal spray product for inactivating avirus comprising: a composition comprising protocatechuic acid andwater; and a mechanical nasal sprayer.
 2. The nasal spray product ofclaim 1, wherein the composition further comprises propylene glycol. 3.The nasal spray product of claim 1, wherein the composition furthercomprises a stabilizer.
 4. The nasal spray product of claim 1, whereinthe stabilizer comprises sodium benzoate.
 5. The nasal spray product ofclaim 1, wherein the stabilizer comprises glycerin.
 6. The nasal sprayproduct of claim 1, wherein the composition comprises protocatechuicacid at a concentration up to 1.24% by weight.
 7. The nasal sprayproduct of claim 1, wherein the composition comprises protocatechuicacid at a concentration between 1.24% and 30% by weight.
 8. A method ofinactivating a virus with the nasal spray product of claim 1 comprising:spraying, coating, or fogging a composition comprising protocatechuicacid, a liquid vehicle comprising water, and a stabilizer, to mucousmembranes of a nose, a mouth, a pharynx, a respiratory tree, or lungs ofa mammal.
 9. The method of claim 8, wherein the composition furthercomprises propylene glycol.
 10. The method of claim 8, wherein the virusis a coronavirus.
 11. The method of claim 8, wherein the virus is aSARS-CoV-2 virus.
 12. The method of claim 8, wherein the compositionfurther comprises a stabilizer.
 13. The method of claim 12, wherein thestabilizer comprises sodium benzoate.
 14. The method of claim 12,wherein the stabilizer comprises glycerin.
 15. The method of claim 8,wherein the protocatechuic acid comprises protocatechuic acid crystals.16. The method of claim 8, wherein the protocatechuic acid crystalsinactivate the virus in the dry state or in solution.
 17. The method ofclaim 8, wherein the virus is inactivated at 95% or greater.
 18. Themethod of claim 8, wherein the virus is inactivated at 99% or greater.19. The method of claim 8, wherein the virus is inactivated at 99.9% orgreater.
 20. The method of claim 8, wherein the composition comprisesprotocatechuic acid at a concentration up to 1.24%.
 21. The method ofclaim 9, wherein the composition comprises protocatechuic acid at aconcentration between 1.24% and 30% by weight.